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WO2003048768A2 - Reseaux - Google Patents

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Publication number
WO2003048768A2
WO2003048768A2 PCT/GB2002/005499 GB0205499W WO03048768A2 WO 2003048768 A2 WO2003048768 A2 WO 2003048768A2 GB 0205499 W GB0205499 W GB 0205499W WO 03048768 A2 WO03048768 A2 WO 03048768A2
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WO
WIPO (PCT)
Prior art keywords
protein
array
proteins
dna
moieties
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2002/005499
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English (en)
Other versions
WO2003048768A3 (fr
Inventor
Jonathan Mark Boutell
Benjamin Leslie James Godber
Darren James Hart
Jonathan David Blackburn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sense Proteomic Ltd
Original Assignee
Sense Proteomic Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sense Proteomic Ltd filed Critical Sense Proteomic Ltd
Priority to EP02788075A priority Critical patent/EP1456668B1/fr
Priority to CA2469403A priority patent/CA2469403C/fr
Priority to AU2002352355A priority patent/AU2002352355B2/en
Priority to DK02788075T priority patent/DK1456668T3/da
Priority to JP2003549912A priority patent/JP4781628B2/ja
Priority to DE60219952T priority patent/DE60219952T2/de
Publication of WO2003048768A2 publication Critical patent/WO2003048768A2/fr
Priority to US10/527,603 priority patent/US20060199220A1/en
Priority to EP03772473A priority patent/EP1543332A2/fr
Priority to PCT/IB2003/005258 priority patent/WO2004025244A2/fr
Priority to AU2003280084A priority patent/AU2003280084A1/en
Publication of WO2003048768A3 publication Critical patent/WO2003048768A3/fr
Anticipated expiration legal-status Critical
Priority to US12/291,466 priority patent/US20090239761A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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    • B01J2219/00722—Nucleotides
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    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
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    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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Definitions

  • SNPs Single nucleotide polymorphisms
  • SNPs More recently 1.42 million SNPs were catalogued by a consortium of researchers in a paper accompanying the human sequence [The International SNP Map Working Group Nature 409 928- 933 (2001)] of which 60,000 were present within genes ('coding' SNPs). Coding SNPs can be further classified according to whether or not they alter the amino acid sequence of the protein and where changes do occur, protein function may be affected resulting in phenotypic variation. Thus there is an unmet need for apparatus and methodology capable of rapidly determining the phenotypes of this large volume of variant sequences.
  • the Inventors herein describe protein arrays and their use to assay, in a parallel fashion, the protein products of highly homologous or related DNA coding sequences.
  • highly homologous or related it is meant those DNA coding sequences which share a common sequence and which differ only by one or more naturally occurring mutations such as single nucleotide polymo ⁇ hisms, deletions or insertions, or those sequences which are considered to be haplotypes (a haplotype being a combination of variations or mutations on a chromosome, usually within the context of a particular gene).
  • Such highly homologous or related DNA coding sequences are generally naturally occurring variants of the same gene.
  • Arrays according to the invention have multiple for example, two or more, individual proteins deposited in a spatially defined pattern on a surface in a form whereby the properties, for example the activity or function of the proteins can be investigated or assayed in parallel by interrogation of the array.
  • Protein arrays according to the invention and their use to assay the phenotypic changes in protein function resulting from mutations differ completely to, and have advantages over, existing DNA based technologies for SNP and other mutational analyses [reviewed in Shi, M.M Clin Chem 47 164- 72 (2001)].
  • These latter technologies include high-throughput sequencing and electrophoretic methods for identifying new SNPs, or diagnostic technologies such as high density oligonucleotide arrays [e.g. Lindblad-Toh, K.
  • Bioinformatics, or computer modelling is possible, especially if a crystal structure is available, but the hypotheses generated still need to be verified experimentally (i.e. through biochemical assay). Frequently though, the role of the mutation remains unclear after bioinformatic or computer-based analysis. Therefore, protein arrays as provided by the invention offer the most powerful route to functional analysis of SNPs.
  • microarray format enables analysis to be carried out using small volumes of potentially expensive ligands • information provided by parallel protein arrays according to the invention will be extremely valuable for drug discovery, pharmacogenomics and diagnostics fields
  • other useful parallel protein arrays may include proteins derived from non-natural (synthetic) mutations of a DNA sequence of interest. Such arrays can be used to investigate interactions between the variant protein thus produced and other proteins, nucleic acid molecules and other molecules, for example ligands or candidate/test small molecules. Suitable methods of carrying out such mutagenesis are described in Current Protocols in Molecular Biology, Volume 1, Chapter 8, Edited by
  • the invention provides a protein array comprising a surface upon which are deposited at spatially defined locations at least two protein moieties characterised in that said protein moieties are those of naturally occurring variants of a DNA sequence of interest.
  • a protein array as defined herein is a spatially defined arrangement of protein moieties in a pattern on a surface.
  • the protein moieties are attached to the surface either directly or indirectly.
  • the attachment can be non-specific (e.g. by physical abso ⁇ tion onto the surface or by formation of a non-specific covalent interaction).
  • the protein moieties are attached to the surface through a common marker moiety appended to each protein moiety.
  • the protein moieties can be inco ⁇ orated into a vesicle or liposome which is tethered to the surface.
  • a surface as defined herein is a flat or contoured area that may or may not be coated/derivatised by chemical treatment.
  • the area can be : a glass slide, one or more beads, for example a magnetised, derivatised and/or labelled bead as known in the art, a polypropylene or polystyrene slide, a polypropylene or polystyrene multi-well plate, a gold, silica or metal object, a membrane made of nitrocellulose, PNDF, nylon or phosphocellulose
  • individual proteins, pairs of proteins or pools of variant proteins may be attached to an individual bead to provide the spatial definition or separation of the array.
  • the beads may then be assayed separately, but in parallel, in a compartmentalised way, for example in the wells of a microtitre plate or in separate test tubes.
  • a protein array comprising a surface according to the invention may subsist as series of separate solid phase surfaces, such as beads carrying different proteins, the array being formed by the spatially defined pattern or arrangement of the separate surfaces in the experiment.
  • the surface coating is capable of resisting non-specific protein abso ⁇ tion.
  • the surface coating can be porous or non-porous in nature.
  • the surface coating provides a specific interaction with the marker moiety on each protein moiety either directly or indirectly (e.g. through a protein or peptide or nucleic acid bound to the surface).
  • SAM2TM membrane Promega, Madison, Wisconsin, USA
  • SAM2TM membrane Promega, Madison, Wisconsin, USA
  • a protein moiety is a protein or a polypeptide encoded by a DNA sequence which is generally a gene or a naturally occurring variant of the gene.
  • the protein moiety may take the form of the encoded protein, or may comprise additional amino acids (not originally encoded by the DNA sequence from which it is derived) to facilitate attachment to the array or analysis in an assay.
  • such proteins may be attached to the array by way of a common feature between the variants.
  • a set of variant proteins may be attached to the array via a binding protein or an antibody which is capable of binding an invariant or common part of the individual proteins in the set.
  • protein moieties according to the invention are proteins tagged (via the combination of the protein encoding DNA sequence with a tag encoding DNA sequence) at either the N- or C- terminus with a marker moiety to facilitate attachment to the array.
  • Each position in the pattern of an array can contain, for example, either:
  • a sample of a single protein type bound to an interacting molecule for example, nucleic acid molecule, antibody, other protein or small molecule.
  • the interacting molecule may itself interact with further molecules.
  • one subunit of an heteromeric protein may be attached to the array and a second subunit or complex of subunits may be tethered to the array via interaction with the attached protein subunit.
  • the second subunit or complex of subunits may then interact with a further molecule, e.g. a candidate drug or an antibody) or
  • protein 1 is derived from a DNA sequence carrying SNP “A” and a 3 base pair deletion "X” whilst “protein 2” is derived from a DNA sequence carrying SNP “A”, SNP “B” and a 3 base pair insertion "Y”.
  • protein 1 is derived from a DNA sequence carrying SNP "A” and a 3 base pair deletion "X” whilst “protein 2” is derived from a DNA sequence carrying SNP “A”, SNP “B” and a 3 base pair insertion "Y”.
  • the protein moiety at each position is substantially pure but in certain circumstances mixtures of between 2 and 100 different protein moieties can be present at each position in the pattern of an array of which at least one is tagged.
  • the proteins derived from the expression of more than one variant DNA sequence may be attached a single position for example, for the pu ⁇ oses of initial bulk screening of a set of variants to determine those sets containing variants of interest.
  • nucleotide polymo ⁇ hisms, deletions or insertions or those sequences which are considered to be haplotypes a haplotype being a combination of variant features on a chromosome, usually within the context of a particular gene).
  • DNA sequences are derived from the same gene in that they map to a common chromosomal locus and encode similar proteins, which may possess different phenotypes.
  • variants are generally naturally occurring versions of the same gene comprising one or more mutations, or their synthetic equivalents, which whilst having different codons, encode the same "wild-type” or variant proteins as those know to occur in a population.
  • DNA molecules having all known mutations in a population are used to produce a set of protein moieties which are attached to the arrays of the invention.
  • the array may comprise a subset of variant proteins derived from DNA molecules possessing a subset of mutations, for example all known germ-line, or inheritable mutations or a subset of clinically relevant or clinically important mutations.
  • Related DNA molecules as defined herein are related by more than just a common tag sequence introduced for the pu ⁇ oses or marking the resulting expressed protein. It is the sequence additional to such tags which is relevant to the relatedness of the DNA molecules.
  • the related sequences are generally the natural coding sequence of a gene and variant forms caused by mutation.
  • the arrays of the invention carry protein moieties which are derived from DNA molecules which differ, i.e. are mutated at 1 to 10, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 discrete locations in the sequence of one DNA molecule relative to another, or more often relative to the wild-type coding sequence (or most common variant in a population).
  • the difference or mutation at each discrete sequence location may be a point mutation such as a base change, for example the substitution of "A" for "G”.
  • a “single nucleotide polymo ⁇ hism” is a mutation of a single nucleotide.
  • the mutation may be a deletion or insertion of 1 to 200, 1 to 100, 1 to 50, 1 to 20 or 1 to 10 bases.
  • insertional mutations are found in "triplet repeat" disorders such as Huntington's Disease - protein variants corresponding to such insertional mutations can be derived from various mutant forms of the gene and attached to the array to permit investigation of their phenotypes.
  • proteins derived from related DNA molecules can be quite different in structure.
  • a related DNA molecule which has undergone a mutation which truncates it, introduces a frame-shift or introduces a stop codon part-way through the wild-type coding sequence may produce a smaller or shorter protein product.
  • mutation may cause the variant protein to have additional structure, for example a repeated domain or a number of additional amino acids either at the termini of the protein or within the sequence of the protein.
  • proteins being derived from related DNA sequences, are included within the scope of the invention.
  • differential pre- translational processing e.g., alternatively spliced transcripts
  • differential post-translational processing e.g. glycosylation occurs at a particular amino acid in one expressed protein, but does not occur in another expressed protein due a codon change in the underlying DNA sequence causing the glycosylated amino acid to be absent.
  • related DNA molecules according to the invention are derived from genes which map to the same chromosomal locus, i.e. the related DNA molecules are different versions of the same protein coding sequence derived from a single copy of a gene, which differ as a result of natural mutation.
  • the wild-type (or the protein encoded by the most common variant DNA sequence in a population) of the protein is preferably included as one of the protein moieties on the array to act as a reference by which the relative activities of the proteins derived from related DNA molecules can be compared.
  • the output of the assay indicates whether the related DNA molecule comprising a mutated gene encodes: (1) a protein with comparable function to the wild-type protein
  • a protein with an activity that can be modified by addition of an extra component e.g. peptide, antibody or small molecule drug candidate.
  • a protein with an altered function under different environmental conditions in the assay for example ionic strength, temperature or pH.
  • the protein moieties of the arrays of the present invention can comprise proteins associated with a disease state, drug metabolism, or may be uncharacterised.
  • the protein moieties encode wild type p53 and allelic variants thereof.
  • the arrays comprises protein moieties which encode a drug metabolising enzyme, preferably wild type p450 and allelic variants thereof.
  • the number of protein variants attached to the arrays of the invention will be determined by the number of variant coding sequences that occur naturally or that are of sufficient experimental, commercial or clinical interest to generate artificially.
  • An array carrying a wild type protein and a single variant would be of use to the investigator.
  • 1 to 10000, 1 to 1000, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 75, 1 to 50, 1 to 25, 1 to 10 or 1 to 5 related DNA molecules are represented by their encoded proteins on an array.
  • p53 the subject of one of the Examples described herein
  • An individual may of course inherit two different germ-line mutations.
  • a p53 variant protein array might carry proteins derived from the 50 germ-line mutations each isolated at a different location, proteins from a clinically relevant subset of 800 somatic coding mutations (where a protein can be expressed) each isolated at a different location (or in groups of 10 at each location) and all possible pair-wise combinations of the 50 germ-line mutations each located at a different location.
  • an array of the invention can usefully represent individual DNA molecules containing more than 1000 different naturally occurring mutations and can accordingly carry many more, for example 10000 or more, separate discrete samples or "spots" of the protein variants derived therefrom either located alone or in combination with other variants.
  • the invention provides a method of making a protein array comprising the steps of a) providing DNA coding sequences which are derived from two or more naturally occurring variants of a DNA sequence of interest b) expressing said coding sequences to provide one or more individual proteins c) purifying said proteins d) depositing said proteins at spatially defined locations on a surface to give an array.
  • Steps c) and d) are preferably combined in a single step. This can be done by means of "surface capture” by which is meant the simultaneous purification and isolation of the protein moiety on the array via the inco ⁇ orated tag as described in the examples below. Furthermore, step c) may be optional as it is not necessary for the protein preparation to be pure at the location of the isolated tagged protein - the tagged protein need not be separated from the crude lysate of the host production cell if purity is not demanded by the assay in which the array takes part.
  • the DNA molecules which are expressed to produce the protein moieties of the array can be generated using techniques known in the art (for example see Current Protocols in Molecular Biology, Volume 1, Chapter 8, Edited by Ausubel, FM, Brent, R, Kingston, RE, Moore, DD, Siedman, JG, Smith, JA, and Struhl, K).
  • SNPs mutations, for example SNPs
  • PCR mutagenesis using the wild-type gene as a template. Therefore, only knowledge of the identity of the mutation, for example SNP (often available in electronic databases), and not the actual mutation containing DNA molecule, is required for protein array fabrication.
  • the wild-type gene encoding the protein of interest, is first cloned into a DNA vector for expression in a suitable host.
  • a suitable host it will be understood by those skilled in the art that the expression host need not be limited to E. coli - yeast, insect or mammalian cells can be used. Use of a eukaryotic host may be desirable where the protein under investigation is known to undergo post-translational modification such as glycosylation.
  • the wild- type gene is mutated to introduce the desired SNPs. The presence of the SNP is confirmed by sequencing following re-cloning.
  • clones can be grown in microtiter plate format (but not exclusively) allowing parallel processing of samples in a format that is convenient for arraying onto slides or plate formats and which provides a high- throughput format.
  • Protein expression is induced and clones are subsequently processed for arraying. This can involve purification of the proteins by affinity chromatography, or preparation of ly sates ready for arraying onto a surface which is selective for the recombinant protein ('surface capture').
  • the DNA molecules may be expressed as fusion proteins to give protein moieties tagged at either the N- or C- terminus with a marker moiety. As described herein, such tags may be used to purify or attach the proteins to the surface or the array.
  • the protein moieties are simultaneously purified from the expression host lysate and attached to the array by means of the marker moiety.
  • the resulting array of proteins can then be used to assay the functions of all proteins in a parallel, and therefore high-throughput manner.
  • the invention provides a method of simultaneously determining the relative properties of members of a set of protein moieties derived from related DNA molecules, comprising the steps of: providing an array as herein described, bringing said array into contact with a test substance, and observing the interaction of the test substance with each set member on the array.
  • the invention provides a method of screening a set of protein moieties derived from related DNA molecules for compounds (for example, a small organic molecule) which restore or disrupt function of a protein, which may reveal compounds with therapeutic advantages or disadvantages for a subset of the population carrying a particular SNP or other mutation.
  • the test substance may be: • a protein for determining relative protein: protein interactions within a set of protein moieties derived from related DNA molecules • a nucleic acid molecule for determining relative protein:DNA or protein:RNA interactions
  • Results obtained from the interrogation of arrays of the invention can be quantitative (e.g. measuring binding or catalytic constants K ⁇ & K M ), semi- quantitative (e.g. normalising amount bound against protein quantity) or qualitative (e.g. functional vs. non-functional).
  • quantitative e.g. measuring binding or catalytic constants K ⁇ & K M
  • semi- quantitative e.g. normalising amount bound against protein quantity
  • qualitative e.g. functional vs. non-functional
  • K ⁇ and B max which describe the affinity of the interaction between ligand and protein and the number of binding sites for that ligand respectively, can be derived from protein array data.
  • quantified or relative amounts of ligand bound to each individual protein spot can be measured at different concentrations of ligand in the assay solution. Assuming a linear relationship between the amount of protein and bound ligand, the (relative) amount of ligand bound to each spot over a range of ligand concentrations used in the assay can be fitted to equation 1, rearrangements or derivations.
  • [L] concentration of ligand used in the assay
  • Figure 1 shows p53 mutant panel expression.
  • E. coli cells containing plasmids encoding human wild type p53 or the indicated mutants were induced for 4h at 30 C.
  • Cells were lysed by the addition of lysozyme and Triton XI 00 and cleared lysates were analysed by Western blot. A band corresponding to full length his- tagged, biotinylated p53 runs at around 70kDa.
  • Figure 2 shows a gel shift assay to demonstrate DNA binding function of E.coli expressed p53.
  • lul of cleared E.coli lysate containing wild type p53 (wt) or the indicated mutant was combined with 250nM DIG-labelled DNA and 0.05mg/ml polydl/dC competitor DNA.
  • the -ve control contained only DNA. Bound and free DNA was separated through a 6% gel (NOVEX), transferred to positively charged membrane (Roche) and DIG-labelled DNA detected using an anti-DIG HRP conjugated antibody (Roche).
  • the DNA:p53 complex is indicated by an arrow.
  • Figure 3 shows microarray data for the p53 DNA binding assay. Lysates were arrayed in a 4x4 pattern onto streptavidin capture membrane as detailed in A) and probed with B) Cy3-labelled anti-histidine antibody or C) Cy3-labelled GADD45 DNA, prior to scanning in an Affymetrix 428 array scanner.
  • Figure 4 shows CKII phosphorylation of p53.
  • 2ul of E.coU lysate containing p53 wild type (wt) or the indicated mutant protein were incubated with or without casein kinase II in a buffer containing ATP for 30min at 30 C.
  • Reactions were Western blotted and phosphorylation at serine 392 detected using a phosphorylation specific antibody.
  • Figure 5 shows microarray data for the CKII phosphorylation assay.
  • the p53 array was incubated with CKII and ATP for lh at 30 C and analysed for phosphorylation at serine 392. Phosphorylation was detected for all proteins on the array except for the truncation mutants Q136X, R196X, R209X, R213X, R306X and for the amino acid mutants L344P and S392A.
  • Figure 6 shows a solution phase MDM2 interaction assay.
  • lOul of p53 containing lysate was incubated with lOul of MDM2 containing lysate and 20ul anti-FLAG agarose in a total volume of 500ul. After incubation for lh at room temperature the anti-FLAG agarose was collected by centrifugation, washed extensively and bound proteins analysed by Western blotting. P53 proteins were detected by Strep/HRP conjugate.
  • FIG 7 shows microarray data for MDM2 interaction.
  • the p53 array was incubated with purified Cy3-labelled MDM2 protein for lh at room temperature and bound MDM2 protein detected using a DNA array scanner (Affymetrix). MDM2 protein bound to all members of the array apart from the W23A and W23G mutants.
  • Figure 8a shows replicate p53 microarrays incubated in the presence of P labelled duplex DNA, corresponding to the sequence of the GADD45 promoter element, at varying concentrations and imaged using a phosphorimager so individual spots could be quantified.
  • Figure 8B shows DNA binding to wild-type p53 (high affinity), R273H (low affinity) and L344P (non-binder) predicting a wild-type affinity of 7 nM.
  • Figure 9A shows a plasmid map of pBJW102.2 for expression of C-terminal BCCP hexa-histidine constructs.
  • Figure 9B shows the DNA sequence of pBJW102.2
  • Figure 9C shows the cloning site of pBJW 102.2 from start codon.
  • Figure 10A shows a vector map of pJW45
  • Figure 10B shows the sequence of the vector pJW45
  • Figure 11 A shows the DNA sequence of Human P450 3A4 open reading frame.
  • Figure 11B shows the amino acid sequence of full length human P450 3A4.
  • Figure 12A shows the DNA sequence of human P450 2C9 open reading frame.
  • Figure 12B shows the amino acid sequence of full length human P450 2C9
  • Figure 13A shows the DNA sequence of human P450 2D6 open reading frame.
  • Figure 13B shows the amino acid sequence of full length human P450 2D6.
  • Figure 14 shows a western blot and coomassie-stained gel of purification of cytochrome P450 3A4 from E. coli.
  • Samples from the purification of cytochrome P450 3A4 were run on SDS-PAGE, stained for protein using coomassie or Western blotted onto nitrocellulose membrane, probed with streptavidin-HRP conjugate and visualised using DAB stain:
  • Lanes 3 Lysed E. coli cells
  • Lanes 4 Supernatant from E. coli cell wash Lanes 5: Pellet from E. coli cell wash
  • Lanes 8 molecular weight markers: 175, 83, 62, 48, 32, 25, 16.5, 6.5 Kda
  • Figure 15 shows the Coomassie stained gel of Ni-NTA column purification of cytochrome P450 3A4. Samples from all stages of column purification were run on SDS-PAGE: Lane 1: Markers 175, 83, 62, 48, 32, 25, 16.5, 6.5 KDa Lane 2: Supernatant from membrane solublisation Lane 3: Column Flow-Through Lane 4: Wash in buffer C Lane 5 : Wash in buffer D
  • Lanes 6&7 Washes in buffer D + 50 mM Imidazole Lanes 8 - 12: Elution in buffer D + 200 mM Imidazole
  • Figure 16 shows the assay of activity for cytochrome P450 2D6 in a reconstitution assay using the substrate AMMC.
  • Recombinant, tagged CYP2D6 was compared with a commercially available CYP2D6 in terms of ability to turnover AMMC after reconstitution in liposomes with NADPH- cytochrome P450 reductase.
  • Figure 17 shows the rates of resorufin formation from BzRes by cumene hydrogen peroxide activated cytochrome P450 3A4. Cytochrome P450 3A4 was assayed in solution with cumene hydrogen peroxide activation in the presence of increasing concentrations of BzRes up to 160 ⁇ M.
  • Figure 18 shows the equilibrium binding of [ Hjketoconazole to immobilised CYP3A4 and CYP2C9.
  • CYP3A4 the data points are the means ⁇ standard deviation, of 4 experiments. Non-specific binding was determined in the presence of lOO ⁇ M ketoconazole (data not shown).
  • Figure 19 shows the chemical activation of tagged, immobilised P450 involving conversion of DBF to fluorescein by CHP activated P450 3A4 immobilised on a streptavidin surface.
  • Figure 20 shows the stability of agarose encapsulated microsomes. Microsomes containing cytochrome P450 2D6 plus NADPH-cytochrome P450 reductase and cytochrome b5 were diluted in agarose and allowed to set in 96 well plates. AMMC turnover was measured immediately and after two and seven days at 4°C.
  • Figure 21 shows the turnover of BzRes by cytochrome P450 3A4 isoforms.
  • Cytochrome P450 3A4 isoforms WT, *1, *2, *3, *4, *5 & *15, (approximately 1 ⁇ g) were incubated in the presence of BzRes (0 - 160 ⁇ M) and cumene hydrogen peroxide (200 ⁇ M) at room temperature in 200 mM KP0 4 buffer pH 7.4. Formation of resorufin was measured over time and rates were calculated from progress curves. Curves describing conventional Michaelis-Menton kinetics were fitted to the data.
  • Figure 22 shows the inhibition of cytochrome P450 3A4 isoforms by ketoconazole.
  • Cytochrome P450 3A4 isoforms WT, *1, *2, *3, *4, *5 & *15, (approximately 1 ⁇ g) were incubated in the presence of BzRes (50 ⁇ M), Cumene hydrogen peroxide (200 ⁇ M) and ketoconazole (0, 0.008, 0.04, 0.2, 1, 5 ⁇ M) at room temperature in 200 mM KP0 4 buffer pH 7.4. Formation of resorufin was measured over time and rates were calculated from progress curves. IC 50 inhibition curves were fitted to the data.
  • Example 1 Use of a protein array for functional analysis of proteins encoded by SNP-containing genes - the p53 protein SNP array
  • tumour suppresser protein p53 Mutations in the tumour suppresser protein p53 have been associated with around 50% of cancers, and more than a thousand SNPs of this gene have been observed. Mutations of the p53 gene in tumour cells (somatic mutation), or in the genome of families with a predisposition to cancer (germline mutation), provide an association between a condition and genotype, but no molecular mechanism. To demonstrate the utility of protein arrays for functional characterisation of coding SNPs, the
  • Inventors have arrayed wild type human p53 together with 46 germline mutations (SNPs). The biochemical activity of these proteins can then be compared rapidly and in parallel using small sample volumes of reagent or ligand.
  • the arrayed proteins are shown to be functional for DNA binding, phosphorylated post-translationally "on-chip" by a known p53 kinase, and can interact with a known p53 -interacting protein, MDM2.
  • SNPs this is the first functional, characterisation of the effect of the mutation on p53 function, and illustrates the usefulness of protein microarrays in analysing biochemical activities in a massively parallel fashion.
  • Wild type p53 cDNA was amplified by PCR from a HeLa cell cDNA library using primers P53F (5' atg gag gag ccg cag tea gat cct ag 3') and P53R (5' gat cgc ggc cgc tea gtc agg ccc ttc tg 3') and ligated into an E.coli expression vector downstream of sequence coding for a poly Histidine-tag and the BCCP domain from the E.coli AccB gene. The ligation mix was transformed into chemically competent XLIBlue cells (Stratagene) according to the manufacturer's instructions. The p53 cDNA sequence was checked by sequencing and found to correspond to wild type p53 protein sequence as contained in the SWISS-PROT entry for p53 [Accession No. P04637].
  • Mutants of p53 were made by using the plasmid containing the wild type p53 sequence as template in an inverse PCR reaction. Primers were designed such that the forward primer was 5' phosphorylated and started with the single nucleotide polymorphism (SNP) at the 5' end, followed by 20-24 nucleotides of p53 sequence. The reverse primer was designed to be complementary to the 20- 24 nucleotides before the SNP. PCR was performed using Pwo polymerase which generated blunt ended products corresponding to the entire p53- containing vector.
  • SNP single nucleotide polymorphism
  • PCR products were gel purified, ligated to form circular plasmids and parental template DNA was digested with restriction endonuclease Dpnl (New England Biolabs) to increase cloning efficiency. Ligated products were transformed into XLIBlue cells, and mutant p53 genes were verified by sequencing for the presence of the desired mutation and the absence of any secondary mutation introduced by PCR.
  • Colonies of XLIBlue cells containing p53 plasmids were inoculated into 2 ml of LB medium containing ampiciUin (70 micrograms /ml) in 48 well blocks (QIAGEN) and grown overnight at 37 °C in a shaking incubator. 40 ⁇ l of overnight culture was used to inoculate another 2 ml of LB/ampicillin in 48 well blocks and grown at 37 °C until an optical density (600nm) of -0.4 was reached. IPTG was then added to 50 ⁇ M and induction continued at 30 °C for 4 hours. Cells were then harvested by centrifugation and cell pellets stored at -80 °C.
  • cell pellets were thawed at room temperature and 40 ⁇ l of p53 buffer (25 mM HEPES pH 7.6, 50 mM KC1, 10% glycerol, 1 mM DTT, 1 mg/ml bovine serum albumin, 0.1% Triton X100) and 10 ⁇ l of 4 mg/ml lysozyme were added and vortexed to resuspend the cell pellet. Lysis was aided by incubation on a rocker at room temperature for 30 min before cell debris was collected by centrifugation at 13000 rpm for 10 min at 4 °C. The cleared supernatant of soluble protein was removed and used immediately or stored at - 20 °C.
  • p53 buffer 25 mM HEPES pH 7.6, 50 mM KC1, 10% glycerol, 1 mM DTT, 1 mg/ml bovine serum albumin, 0.1% Triton X100
  • Soluble protein samples were boiled in SDS containing buffer for 5 min prior to loading on 4-20% Tris-Glycine gels (NOVEX) and run at 200 V for 45 min. Protein was transferred onto PVDF membrane (Hybond-P, Amersham) and probed for the presence of various epitopes using standard techniques. For detection of the histidine-tag, membranes were blocked in 5% Marvel /PBST and anti-RGSHis antibody (QIAGEN) was used as the primary antibody at 1/1000 dilution.
  • DNA binding function of expressed p53 was assayed using a conventional gel shift assay. Oligos DIGGADD45A (5'DIG-gta cag aac atg tct aag cat get ggg gac-3') and GADD45B (gtc ccc age atg ctt aga cat gtt ctg tac 3') were annealed together to give a final concentration of 25 ⁇ M dsDNA. Binding reactions were assembled containing 1 ⁇ l of cleared lysate, 0.2 ⁇ l of annealed DIG-labelled GADD45 oligos and 1 ⁇ l of polydl/dC competitor DNA (Sigma) in 20 ⁇ l of p53 buffer.
  • DIGGADD45A 5'DIG-gta cag aac atg tct aag cat get ggg gac-3'
  • GADD45B gtc cc age atg ctt aga cat
  • Phosphorylation reactions contained 2 ⁇ l of p53 lysate, 10 mM MgCl 2 , 100 ⁇ M ATP and 0.1U of CKII in 20 ⁇ l of p53 buffer. Reactions were incubated at 30 °C for 30 min, reaction products separated through 4-20% NOVEX gels and transferred onto PVDF membrane. Phosphorylation of p53 was detected using an antibody specific for phosphorylation of p53 at serine 392 (Cell Signalling Technology), used at 1/1000 dilution in Marvel/TBST. Secondary antibody was an anti-rabbit HRP conjugate (Cell Signalling Technology), used at 1/2000 dilution. MDM2 interaction assay
  • the cDNA for the N-terminal portion of MDM2 (amino acids 17-127) was amplified from a cDNA library and cloned downstream of sequences coding for a His-tag and a FLAG-tag in an E. coli expression vector. Plasmids were checked by sequencing for correct MDM2 sequence and induction of E. coli cultures showed expression of a His and FLAG tagged soluble protein of the expected size.
  • binding reactions were assembled containing lO ⁇ l p53 containing lysate, lO ⁇ l MDM2 containing lysate, 20 ⁇ l anti-FLAG agarose in 500 ⁇ l phosphate buffered saline containing 300mM NaCl, 0.1% Tween20 and 1% (w/v) bovine serum albumin. Reactions were incubated on a rocker at room temperature for 1 hour and FLAG bound complexes harvested by centrifugation at 5000 ⁇ m for 2min. After extensive washing in PBST, FLAG bound complexes were denatured in SDS sample buffer and Western blotted. Presence of biotinylated p53 was detected by Streptavidin/HRP conjugate.
  • l ⁇ l of purified Cy3 labelled MDM2 protein was incubated with the arrays in 500 ⁇ l PBS/300mM NaCl/0.1% Tween20/1% BSA for lh at room temperature. After washing for 3 x 5min in the same buffer, arrays were dried, mounted onto glass slides and analysed for Cy3 fluorescence as for the DNA binding assay.
  • the full length p53 open reading frame was amplified from a Hela cell cDNA library by PCR and cloned downstream of the tac promoter in vector pQE80L into which the BCCP domain from the E.coli gene ACCB had already been cloned.
  • the resultant p53 would then be His and biotin tagged at its N-terminus, and figure 1 shows Western blot analysis of soluble protein from induced E.coli cultures. There is a clear signal for His-tagged, biotinylated protein at around 66kDa, and a band of the same size is detected by the p53 specific antibody pAbl ⁇ Ol (data not shown).
  • the plasmid encoding this protein was fully sequenced and shown to be wild type p53 cDNA sequence.
  • This plasmid was used as the template to construct the mutant panel, and figure 1 also shows analysis of the expression of a selection of those mutants, showing full length protein as expected for the single nucleotide polymo ⁇ hisms, and truncated proteins where the mutation codes for a STOP codon.
  • the mutants were also sequenced to confirm presence of the desired mutation and absence of any secondary mutations.
  • affinity tags eg FLAG, myc, VSV
  • an expression host other than E. coli can be used (eg. yeast, insect cells, mammalian cells) if required.
  • this array was focussed on the naturally occurring germline SNPs of p53, other embodiments are not necessarily restricted to naturally occurring SNPs ("synthetic" mutants) or versions of the wild type protein which contain more than one SNP. Other embodiments can contain versions of the protein which are deleted from either or both ends (a nested-set). Such arrays would be useful in mapping protein:ligand interactions and delineating functional domains of unknown proteins.
  • E. coli expressed p53 is functional for DNA binding
  • FIG. 2 shows an example result from these gel shift assays, showing DNA binding by wild type p53 as well as mutants R72P, P82L and R181C. The first 2 mutants would still be expected to bind DNA as these mutations are outside of the DNA binding domain of p53. Having demonstrated DNA binding using a conventional gel based assay, the Inventors then wanted to show the same function for p53 arrayed on a surface.
  • Figure 3C shows the result of binding Cy3-labelled DNA to the p53 mutant panel arrayed onto SAM2TM membrane (Promega, Madison, Wisconsin, USA).
  • SAM2TM membrane in this example of a SNP array
  • other surfaces which can be used for arraying proteins onto include but are not restricted to glass, polypropylene, polystyrene, gold or silica slides, polypropylene or polystyrene multi-well plates, or other porous surfaces such as nitrocellulose, PVDF and nylon membranes.
  • the SAM2TM membrane specifically captures biotinylated molecules and so purifies the biotinylated p53 proteins from the mutant panel cell lysates. After washing unbound DNA from the array, bound DNA was visualised using an Affymetrix DNA array scanner.
  • Lanes 3 and 4 show the corresponding results for S392A, which as expected only shows background signal for phosphorylation by CKII. This assay was then applied in a microarray format, which as can be seen from figure 5 shows phosphorylation for all of the mutant panel except the S392A mutant and those mutants which are truncated before residue 392.
  • the Inventors have used a novel protein chip technology to characterise the effect of 46 germline mutations on human p53 protein function.
  • the arrayed proteins can be detected by both a His-tagged antibody and also a p53 specific antibody. This array can be used to screen for mutation specific antibodies which could have implications for p53 status diagnosis.
  • the Inventors were able to demonstrate functionality of the wild type protein by conventional gel based assays, and have achieved similar results performing the assays in a microarray format. Indeed, for a DNA binding assay the microarray assay appeared to be more sensitive than the conventional gel shift assay.
  • These arrays can be stored at -20 C in 50% glycerol and have been shown to still be functional for DNA binding after 1 month (data not shown).
  • the CKII phosphorylation assay results are as expected, with phosphorylation being detected for all proteins which contained the serine at residue 392. This analysis can obviously be extended to a screen for kinases that phosphorylate p53, or for instance for kinases that differentially phosphorylate some mutants and not others, which could themselves represent potential targets in cancer.
  • the MDM2 interaction assay again shows the validity of the protein array format, with results for wild type and the p53 mutants mirroring those obtained using a more conventional pull down assay. These results also show that our protein arrays can be used to detect protein :protein interactions. Potentially these arrays can be used to obtain quantitative binding data (ie K D values) for protei protein interactions in a high-throughput manner not possible using current methodology.
  • Example 2 Quantitative DNA binding on the p53 protein microarray
  • Oligonucleotides with the GADD45 promoter element sequence (5'-gta cag aac atg tct aag cat get ggg gac-3' and 5 '-gtc ccc age atg ctt aga cat gtt ctg tac-3') were radiolabeUed with gamma 33 P-ATP (Amersham Biosciences, Buckinghamshire, UK) and T4 kinase (Invitrogen, Carlsbad, CA), annealed in p53 buffer and then purified using a Nucleotide Extraction column (Qiagen, Valencia, CA).
  • Proteins with near wild-type affinity for DNA generally had mutations located outside of the DNA-binding domain and include R72P, P82L, R306P and G325V.
  • R337C is known to affect the oligomerisation state of p53 but at the assay temperature used here it is thought to be largely tetrameric [Davison, T.S., Yin, P., Nie, E., Kay, C. & Arrowsmith, CH. Characterisation of the oligomerisation defects of two p53 mutants found in families with Li-Fraumeni and Li-Fraumeni like syndrome. Oncogene 17, 651-656 (1998).], consistent with the affinity measured here.
  • mutants fall into a group displaying considerably reduced specific activities, apparent from very low B max values, even when normalised according to the amount of protein present in the relevant spot.
  • DNA binding was compromised to such a level that although binding was observed, it was not accurately quantifiable due to low signal to background ratios e.g. P151S and G245C.
  • low signal intensities yielded measurable K_ values, but with wide confidence limits.
  • Phase 1 DME's include the Cytochrome p450's and the Flavin mono oxygenases (FMO's) and the Phase 2 DME's, UDP-glycosyltransferase (UGTs), glutathione S transferases (GSTs), sulfotransferases (SULTs), N -acetyltransferases (NATs), drug binding nuclear receptors and drug transporter proteins.
  • FMO's Flavin mono oxygenases
  • Phase 2 DME's Phase 2 DME's
  • UDP-glycosyltransferase UDP-glycosyltransferase
  • GSTs glutathione S transferases
  • SULTs sulfotransferases
  • NATs N -acetyltransferases
  • the full complement, or a significant proportion of human DMEs are present on the arrays of the invention.
  • Such an array can include (numbers in parenthesis currently described in the Swiss Prot database): all the human P450s (119), FMOs (5), UDP-glycosyltransferase (UGTs) (18), GSTs (20), sulfotransferases (SULTs) (6), N-acetyltransferases (NATs) (2), drug binding nuclear receptors (33) and drug transporter proteins (6).
  • This protein list does not include those yet to be characterised from the human genome sequencing project, splice variants known to occur for the P450s that can switch substrate specificity or polymo ⁇ hisms known to affect the function and substrate specificity of both the P450s and the phase 2 DMEs.
  • the human cytochrome p450s have a conserved region at the N-terminus, this includes a hydrophobic region which faciliates lipid association, an acidic or 'stop transfer' region, which stops the protein being fed further into the membrane, and a partially conserved proline repeat. Three versions of the p450s were produced with deletions up to these domains, the N-terminal deletions are shown below.
  • the human CYP2D6 was amplified by PCR from a pool of brain, heart and liver cDNA libraries (Clontech) using specific forward and reverse primers (T017F and T017R). The PCR products were cloned into the pMD004 expression vector, in frame with the N-terminal His-BCCP tag and using the Notl restriction site present in the reverse primer.
  • primers were used which inco ⁇ orated an Sfil cloning site at the 5' end and removed the stop codon at the 3' to allow in frame fusion with the C-terminal tag.
  • the primers T017CR together with either T017CF1, T017CF2, or T017CF3 allowed the deletion of 29, 18 and 0 amino acids from the N-terminus of CYP2D6 respectively.
  • Primer sequences are as follows:
  • T017F 5 ' -GCTGCACGCTACCCACCAGGCCCCCTG-3 ' .
  • T017R 5 ' -TTGCGGCCGCTCTTCTACTAGCGGGGCACAGCACAAAGCTCATAG-3 '
  • T017CF1 5 ' -TATTCTCACTGGCCATTACGGCCGCTGCACGCTACCCACCAGGCCCCCTG-3 '
  • T017CF2 5'-
  • PCR products were resolved by agarose gel electrophoresis, those products of the correct size were excised from the gel and subsequently purified using a gel extraction kit. Purified PCR products were then digested with either Sfil or Notl and ligated into the prepared vector backbone (Fig. 9C). Correct recombinant clones were determined by PCR screening of bacterial cultures, Western blotting and by DNA sequence analysis.
  • CYP3A4 and CYP2C9 were cloned from cDNA libraries by a methodology similar to that of CYP2D6.
  • Primer sequences to amplify CYP3A4 and CYP2C9 for cloning into the N-terminal vectors are as follows; 2C9
  • T015F 5 ' -CTCCCTCCTGGCCCCACTCCTCTCCCAA-3 '
  • T015R 5 ' -TTTGCGGCCGCTCTTCTATCAGACAGGAATGAAGCACAGCCTGGTA-3 '
  • 3A4 TO09F 5 ' -CTTGGAATTCCAGGGCCCACACCTCTG-3 '
  • 3A4 T009CF1 5 ' -TATTCTCACTGGCCATTACGGCCTATGGAACCCATTCACATGGACTTTTTA
  • T015CF1 5 ' -TATTCTCACTGGCCATTACGGCCAGACAGAGCTCTGGGAGAGGAAAACTCCCTC CTGGCCCCACTCCTCTCCCAG-3 '
  • T015CF2 5 ' -TATTCTCACTGGCCATTACGGCCCTCCCTCCTGGCCCCACTCCTCTCCCAG-3 '
  • T015CR 5 ' -GACAGGAATGAAGCACAGCTGGTAGAAGG-3 '
  • the full length or Hydrophobic peptide (C3) version of 2C9 was produced by inverse PCR using the 2C9-stop transfer clone (CI) as the template and the following primers:
  • NADPH-cytochrome P450 reductase was amplified from fetal liver cDNA (Clontech), the PCR primers [NADPH reductase FI 5'- GGATCGACATATGGGAGACTCCCACGTGGACAC-3'; NADPH reductase R 1 5 ' -CCGATA AGCTT ATC AGCTCCAC ACGTCC AGGG AG-3 ' ] inco ⁇ orated a Nde I site at 5' and a Hind III site at the 3' of the gene to allow cloning.
  • the PCR product was cloned into the pJW45 expression vector (Fig. 10A&B)), two stop codons were included on the reverse primer to ensure that the His-tag was not translated. Correct recombinant clones were determined by PCR screening of bacterial cultures, and by sequencing.
  • Example 5 Cloning of polymo ⁇ hic variants of H. sapiens cytochrome P450s CYP2C9. CYP2D6 and CYP3A4
  • CYP2C9*7R 5 ' -CACCACGTGCTCCAGGTCTCTA-3 ' CYP2D6*10AF1 :
  • CYP2D6 *17R 5 ' - GATGGGCACAGGCGGGCGGTC-3 '
  • CYP2D6 *9F 5 ' -GCCAAGGGGAACCCTGAGAGC-3 '
  • CYP2D6 * 9R 5 ' -CTCCATCTCTGCCAGGAAGGC-3 '
  • CYP3A4 *2F 5 ' -CCAATAACAGTCTTTCCATTCCTC-3
  • CYP3A4 *2R 5 ' -GAGAAAGAATGGATCCAAAAAATC-3
  • E. coli XL- 10 gold (Stratagene) was used as a host for expression cultures of P450 3A4. Starter cultures were grown overnight in LB media supplemented with lOOmg per litre ampiciUin. 0.5 litre Terrific Broth media plus lOOmg per litre ampiciUin and ImM thiamine and trace elements were inoculated with 1/100 dilution of the overnight starter cultures. The flasks were shaken at 37 °C until cell density OD 60 o was 0.4 then ⁇ -Aminolevulinic acid (ALA) was added to the cells at 0.5mM for 20 min at 30°C. The cells were supplemented with 50 ⁇ M biotin then induced with optimum concentration of IPTG (30- lOO ⁇ M) then shaken overnight at 30°C.
  • ALA ⁇ -Aminolevulinic acid
  • the E. coli cells from 0.5 litre cultures were divided into 50 ml aliquots, cells pelleted by centrifugation and cell pellets stored at -20°C. Cells from each pellet were lysed by resuspending in 5ml buffer A (lOOmM Tris buffer pH 8.0 containing 100 mM EDTA, lOmM ⁇ -mercaptoethanol, lOx stock of Protease inhibitor cocktail- Roche 1836170, 0.2mg/ml Lysozyme). After 15 minutes incubation on ice 40 ml of ice-cold deionised water was added to each resuspended cell pellet and mixed.
  • 5ml buffer A lOOmM Tris buffer pH 8.0 containing 100 mM EDTA, lOmM ⁇ -mercaptoethanol, lOx stock of Protease inhibitor cocktail- Roche 1836170, 0.2mg/ml Lysozyme
  • Membrane associated protein was then solubilised by the addition of 2 ml buffer C (50mM potassium phosphate pH 7.4, lOx stock of Protease inhibitor cocktail- Roche 1836170, 10 mM ⁇ -mercaptoethanol, 0.5 M NaCl and 0.3% (v/v) Igepal CA-630) and incubating on ice with gentle agitation for 30 minutes before centrifugation at 10,000g for 15 min at 4°C and the supernatant (Fig. 14) was then applied to Talon resin (Clontech).
  • 2 ml buffer C 50mM potassium phosphate pH 7.4, lOx stock of Protease inhibitor cocktail- Roche 1836170, 10 mM ⁇ -mercaptoethanol, 0.5 M NaCl and 0.3% (v/v) Igepal CA-630
  • a 0.5 ml column of Ni-NTA agarose (Qiagen) was poured in disposable gravity columns and equilibrated with 5 column volumes of buffer C. Supernatant was applied to the column after which the column was successively washed with 4 column volumes of buffer C, 4 column volumes of buffer D (50mM potassium phosphate pH 7.4, lOx stock of Protease inhibitor cocktail- Roche 1836170, 10 mM ⁇ -mercaptoethanol, 0.5 M NaCl and 20% (v/v) Glycerol) and 4 column volumes of buffer D + 50 mM Imidazole before elution in 4 column volumes of buffer D + 200 mM Imidazole (Fig. 15). 0.5ml fractions were collected and protein containing fractions were pooled aliquoted and stored at -80°C.
  • Purified P450s were diluted to a concentration of 0.2 mg / ml in 20 mM potassium phosphate (pH 7.4) in the presence and absence of 10 mM KCN and an absorbance scan measured from 600 - 260 nm. The percentage bound heme
  • Example 8 Reconstitution and assay of cytochrome P450 enzymes into liposomes with NADPH-cytochrome P450 reductase
  • Liposomes are prepared by dissolving a 1:1:1 mixture of 1,2-dilauroyl-sn- glycero-3-phosphocholine, 1 ,2-dileoyl-sn-glycero-3-phosphocholine, 1 ,2- dilauroyl-sn-glycero-3-phosphoserine in chloroform, evaporating to dryness and subsequently resuspending in 20 mM potassium phosphate pH 7.4 at 10 mg/ml.
  • liposomes 4 ⁇ g of liposomes are added to a mixture of purified P450 2D6 (20 pmol), NADPH P450 reductase (40 pmol), cytochrome b5 (20 pmol) in a total volume of 10 ⁇ l and preincubated for 10 minutes at 37°C.
  • the liposomes are diluted to 100 ⁇ l in assay buffer in a black 96 well plate, containing HEPES / KOH (pH 7.4, 50 mM), NADP+ (2.6 mM), glucose-6-phosphate (6.6 mM), MgCl 2 (6.6 mM) and glucose-6-phosphate dehyrogenase (0.4 units / ml).
  • Assay buffer also contains an appropriate fluorogenic substrate for the cytochrome P450 isoform to be assayed: for P450 2D6 AMMC, for P450 3A4 dibenzyl fluorescein (DBF) or resorufin benzyl ether (BzRes) can be used and for 2C9 dibenzyl fluorescein (DBF).
  • the reactions are stopped by the addition of 'stopping solution' (80% acetonitrile buffered with Tris) and products are read using the appropriate wavelength filter sets in a fluorescent plate reader (Fig. 16).
  • P450s can also be activated chemically by, for example, the addition of 200 ⁇ M cumene hydroperoxide in place of the both the co-enzymes and regeneration solution (Fig. 17).
  • fluorescently measured rates of turnover can be measured in the presence of inhibitors.
  • CYP3A4 (lO ⁇ g/ml in 50mM HEPES/0.01% CHAPS, pH 7.4) was placed in streptavidin immobiliser plates (Exiqon) (lOO ⁇ l per well) and shaken on ice for 1 hour. The wells were aspirated and washed twice with 50mM HEPES/0.01% CHAPS. [ 3 H] -ketoconazole binding to immobilised protein was determined directly by scintillation counting. Saturation experiments were performed using [ 3 H] ketoconazole (5Ci/mmol, American Radiochemicals Inc., St. Louis) in 50mM HEPES pH 7.4, 0.01% CHAPS and 10% Superblock (Pierce) ( Figure 18).
  • CYP3A4 was immobilised in streptavidin immobiliser plates as described in Example 9 and was then incubated with dibenzyl fluorescein and varying concentrations (0-300 ⁇ M) of cumene hydrogen peroxide. End point assays demonstrated that the tagged, immobilised CYP3A4 was functional in a turnover assay with chemical activation (Fig. 19).
  • Example 11 Immobilisation of P450s through gel encapsulation of liposomes or microsomes
  • cytochrome P450 enzymes After reconstitution of cytochrome P450 enzymes together with NADPH- cytochrome P450 reductase in liposomes or microsomes, these can then be immobilised on to a surface by encapsulation within a gel matrix such as agarose, polyurethane or polyacrylamide.
  • a gel matrix such as agarose, polyurethane or polyacrylamide.
  • LMT low melting temperature
  • cytochrome P450 3A4, cytochrome b5 and NADPH-cytochrome P450 reductase were then diluted into the LMT agarose such that 50 ⁇ l of agarose contained 20, 40 and 20 pmol of P450 3A4, NADPH-cytochrome P450 reductase and cytochrome b5 respectively.
  • 50 ⁇ l of agarose-microsomes was then added to each well of a black 96 well microtitre plate and allowed to solidify at room temperature.
  • the activity of the immobilised P450s was assessed over a period of 7 days (Fig. 20). Aliquots of the same protein preparation stored under identical conditions, except that they were not gel-encapsulated, were also assayed over the same period, which revealed that the gel encapsualtion confers significant stability to the P450 activity.
  • Example 12 Quantitative determination of affect of 3A4 polymorphisms on activity
  • cytochrome P450 3A4 isoforms *1, *2, *3, *4, *5 & *15 were incubated in the presence of BzRes and cumene hydrogen peroxide (200 ⁇ M) in the absence and presence of ketoconazole at room temperature in 200 mM KP0 4 buffer pH 7.4 in a total volume of 100 ⁇ l in a 96 well black microtitre plate. A minimum of duplicates were performed for each concentration of BzRes or ketoconazole. Resorufin formation of was measured over time by the increase in fluorescence (520 nm and 580 nm excitation and emission filters respectively) and initial rates were calculated from progress curves (Fig. 21).
  • V V max / (l + (K M / S)) where V and S are initial rate and substrate concentration respectively. V max values were then normalised for cytochrome P450 concentration and scaled to the wild-type enzyme (Table 7).
  • Example 13 Array-based assay of immobilised CYP3A4 polymorphisms
  • Cytochrome P450 polymo ⁇ hisms can be assayed in parallel using an array format to identify subtle differences in activity with specific small molecules.
  • purified cytochrome P450 3A4 isoforms *1, *2, *3, *4, *5 & *15 . can be individually reconstituted in to liposomes with NADPH-cytochrome P450 reductase as described in Example 11.
  • the resultant liposomes preparation can then be diluted into LMP agarose and immobilised into individual wells of a black 96 well microtitre plate as described in Example 11.
  • the immobilised proteins can then be assay ed as described in Example 11 by adding lOO ⁇ l of assay buffer containing BzRes +/- ketoconazole to each well.
  • Chemical activation (as described in Example 12) can also be used in an array format.
  • the Inventors have developed a novel protein array technology for massively parallel, high-throughout screening of SNPs for the biochemical activity of the encoded proteins. Its applicability was demonstrated through the analysis of various functions of wild type p53 and 46 SNP versions of p53 as well as with allelic variants of p450. The same surface and assay detection methodologies can now be applied to other more diverse arrays currently being developed. Due to the small size of the collection of proteins being studied here, the spot density of our arrays was relatively small, and each protein was spotted in quadruplicate. Using current robotic spotting capabilities it is possible to increase spot density to include over 10,000 proteins per array.

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Abstract

L'invention concerne des réseaux de protéines et l'utilisation de ceux-ci pour tester en parallèle des produits protéiques de séquences de codage d'ADN hautement homologues ou relatives. Hautement homologues ou relatives signifie que ces séquences de codage d'ADN partagent une séquence commune et diffèrent seulement d'une ou de plusieurs mutations d'origine naturelle, telles que des polymorphismes nucléotidiques uniques, des délétions ou des introductions, ou que ces séquences sont considérées comme étant des haplotypes (soit une combinaison de variations ou de mutations sur un chromosome, généralement pour un gène particulier). Ces séquences de codage d'ADN hautement homologues ou relatives sont généralement des variants du même gène d'origine naturelle. Des réseaux selon l'invention présentant plusieurs, par exemple, au moins deux, protéines individuelles déposées, selon un motif défini dans l'espace, sur une surface, sous une forme permettant d'effectuer des recherches relatives aux propriétés, par exemple, l'activité ou une fonction des protéines, ou de tester celles-ci en parallèle, par interrogation du réseau,.
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AU2002352355A AU2002352355B2 (en) 2001-12-05 2002-12-05 Protein arrays for allelic variants and uses thereof
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Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1543332A2 (fr) * 2002-09-16 2005-06-22 Sense Proteomic Limited Reseaux de proteines et leurs utilisations
EP1561823A1 (fr) * 2004-02-04 2005-08-10 Biotez Berlin-Buch GmbH Procédé pour la détection des polymorphismes de nucléotide simple (SNP) des gênes du métabolisme des médicaments et dispositif pour l'utilisation correspondante
WO2006002525A1 (fr) * 2004-06-30 2006-01-12 Tm Bioscience Pgx, Inc. Methode de detection de mutations dans le gene codant pour le cytochrome p450-2c9
JP2007510404A (ja) * 2003-10-15 2007-04-26 ジェネッサンス ファーマシューティカルズ，インコーポレイティド アルツハイマー病の発症年齢に関連するｎｔｒｋ１遺伝子マーカー
JP2007514417A (ja) * 2003-11-24 2007-06-07 ジェネッサンス ファーマシューティカルズ，インコーポレイティド アルツハイマー病の進行に関連するｎｔｒｋ１遺伝子マーカー
WO2008123948A1 (fr) 2007-03-27 2008-10-16 Vermillion, Inc. Biomarqueurs pour cancer des ovaires
WO2009114292A1 (fr) 2008-03-10 2009-09-17 Lineagen, Inc. Signatures de biomarqueur de copd
WO2012051519A2 (fr) 2010-10-14 2012-04-19 The Johns Hopkins University Biomarqueurs d'une lésion cérébrale
EP2469279A1 (fr) 2006-03-11 2012-06-27 The Board Of Trustees Of The Leland Stanford Junior University Cystatin C, lysozyme et bêta-2 microglobuline en tant que biomarqueur d'une maladie des artères périphériques
WO2012155134A2 (fr) 2011-05-12 2012-11-15 The Johns Hopkins University Réactifs de dosage pour kit de diagnostic de la neurogranine
WO2013106886A1 (fr) 2012-01-20 2013-07-25 Adelaide Research & Innovation Pty Ltd Biomarqueurs pour le cancer gastrique et leurs utilisations
WO2014100852A1 (fr) 2012-12-24 2014-07-03 Royal Melbourne Institute Of Technology Inhibition de la croissance du cancer et de métastase
WO2014182896A1 (fr) 2013-05-10 2014-11-13 Johns Hopkins University Compositions présentant une grande spécificité pour l'évaluation du cancer de l'ovaire
US9228223B2 (en) 2007-02-12 2016-01-05 Codexis, Inc. Structure-activity relationships
EP2993474A1 (fr) 2005-06-24 2016-03-09 Vermillion, Inc. Biomarqueurs pour le cancer de l'ovaire: la beta-2-microglobuline
WO2017099829A1 (fr) 2015-12-11 2017-06-15 The General Hospital Corporation Compositions et procédés de traitement de glioblastome multiforme résistant aux médicaments
WO2019035005A1 (fr) 2017-08-16 2019-02-21 Medimmune, Llc Compositions et méthodes pour le traitement de la dermatite atopique et la sélection du traitement
US10222386B2 (en) 2014-09-19 2019-03-05 The Johns Hopkins University Biomarkers of congnitive dysfunction
WO2019236632A1 (fr) 2018-06-04 2019-12-12 Avon Products. Inc. Biomarqueurs protéiques permettant d'identifier et de traiter une peau vieillissante et des affections cutanées
EP3757226A2 (fr) 2013-07-17 2020-12-30 The Johns Hopkins University Dosage de biomarqueurs multiprotéiniques pour la détection et l'issue de lésions cérébrales
US11041865B2 (en) 2015-02-20 2021-06-22 The Johns Hopkins University Biomarkers of myocardial injury
WO2022072479A1 (fr) 2020-09-29 2022-04-07 The Johns Hopkins University Biomarqueurs protéomiques intégrés destinés à la détection d'un cancer agressif de la prostate
WO2023122723A1 (fr) 2021-12-23 2023-06-29 The Broad Institute, Inc. Panels et procédés de diagnostic et de traitement du cancer du poumon
US12116637B2 (en) 2020-07-24 2024-10-15 The Regents Of The University Of Michigan Compositions and methods for detecting and treating high grade subtypes of uterine cancer
WO2025106804A1 (fr) 2023-11-16 2025-05-22 Massachusetts Institute Of Technology Compositions et méthodes permettant la caractérisation d'un stress hépatocytaire
WO2025217069A1 (fr) 2024-04-08 2025-10-16 Beth Israel Deaconess Medical Center, Inc. Compositions et méthodes de traitement de la cardiotoxicité

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7094568B2 (en) * 2000-08-17 2006-08-22 Sense Proteomic Ltd. Method for producing proteins tagged at the N- or C-terminus
US7297553B2 (en) 2002-05-28 2007-11-20 Nanosphere, Inc. Method for attachment of silylated molecules to glass surfaces
AU2002322458A1 (en) * 2001-07-13 2003-01-29 Nanosphere, Inc. Method for immobilizing molecules onto surfaces
GB0202018D0 (en) * 2002-01-29 2002-03-13 Sense Proteomic Ltd Tag and method
US20060275855A1 (en) * 2002-02-21 2006-12-07 Blackburn Jonathan M Enzyme array and assay
GB0205910D0 (en) * 2002-03-13 2002-04-24 Sense Proteomic Ltd Arrays and methods
US20030228709A1 (en) * 2002-03-25 2003-12-11 Kozlowski Roland Zbignieiw Arrays
EP1561109A1 (fr) * 2002-09-03 2005-08-10 Zeptosens AG Plate-forme analytique et procede d'identification avec des substances d'analyse a identifier dans un echantillon, se presentant sous forme de partenaires de liaison specifiques immobilises
CN111480079A (zh) * 2017-10-16 2020-07-31 伊纳诺生物股份有限公司 疾病蛋白质组蛋白质阵列及其应用

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3179481B2 (ja) 1990-06-27 2001-06-25 プリンストン ユニバーシティ 突然変異体ｐ５３検出用プローブ
US5412087A (en) * 1992-04-24 1995-05-02 Affymax Technologies N.V. Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces
US5912120A (en) * 1992-04-09 1999-06-15 The United States Of America As Represented By The Department Of Health And Human Services, Cloning, expression and diagnosis of human cytochrome P450 2C19: the principal determinant of s-mephenytoin metabolism
JPH06213660A (ja) * 1993-01-19 1994-08-05 Aisin Seiki Co Ltd 像の近似直線の検出方法
US5986054A (en) * 1995-04-28 1999-11-16 The Hospital For Sick Children, Hsc Research And Development Limited Partnership Genetic sequences and proteins related to alzheimer's disease
FR2775984B1 (fr) * 1998-03-11 2006-09-15 Bioscreen Therapeutics Sa Criblage differentiel qualitatif
US6406921B1 (en) * 1998-07-14 2002-06-18 Zyomyx, Incorporated Protein arrays for high-throughput screening
US6576478B1 (en) 1998-07-14 2003-06-10 Zyomyx, Inc. Microdevices for high-throughput screening of biomolecules
US6197599B1 (en) * 1998-07-30 2001-03-06 Guorong Chin Method to detect proteins
US6692908B1 (en) * 1998-11-05 2004-02-17 Stanford University Prevention and treatment of HCV infection employing antibodies that inhibit the interaction of HCV virions with their receptor
US20010055764A1 (en) * 1999-05-07 2001-12-27 Empedocles Stephen A. Microarray methods utilizing semiconductor nanocrystals
EP1188058B1 (fr) * 1999-06-18 2005-05-11 Biacore AB Procede et appareil de dosage biologique d'une drogue candidate destines a evaluer un parametre pharmacocinetique associe
EP1222274A2 (fr) * 1999-10-19 2002-07-17 Ludwig Institute For Cancer Research Peptides antigeniques mage-a12 et utilisation
EP1203238B1 (fr) * 2000-01-31 2010-08-11 Sense Proteomic Limited Procedes de preparation de reseaux de proteines exprimees et leur utilisation en criblage rapide
US7816098B2 (en) * 2000-01-31 2010-10-19 Sense Proteomic Limited Methods of making and using a protein array
DE60142765D1 (de) 2000-01-31 2010-09-23 Sense Proteomic Ltd Verfahren zur herstellung von protein expression arrays und anwendung derselben in schnellem screening
JP2003522962A (ja) 2000-02-16 2003-07-29 クァンタム・ドット・コーポレイション 半導体ナノクリスタルを使用するマイクロアレイ法
KR20040010041A (ko) * 2000-05-04 2004-01-31 예일 유니버시티 단백질 활성의 스크리닝을 위한 고밀도 단백질 어레이
US7094568B2 (en) * 2000-08-17 2006-08-22 Sense Proteomic Ltd. Method for producing proteins tagged at the N- or C-terminus
AU2002248256B2 (en) * 2000-12-26 2006-09-28 Applied Molecular Evolution, Inc. Butyrylcholinesterase polypeptide variants with increased catalytic efficiency and methods of use
ATE365229T1 (de) * 2000-12-28 2007-07-15 Epidauros Biotechnologie Ag Identifizierung von genetischen determinanten der polymorphen cyp3a5 expression
CA2438267C (fr) 2001-02-20 2012-05-15 Larry E. Morrison Procedes et sondes servant a detecter le cancer
EP2290055A3 (fr) * 2001-06-01 2011-06-01 PGXHealth, LLC Polymorphismes dans le gene humain du polypeptide 2c8 du cytochrome p450 et leur utilisation dans des applications diagnostiques et therapeutiques
EP2287330A1 (fr) * 2001-11-01 2011-02-23 Rensselaer Polytechnic Institute Jeux ordonnés de microéchantillons solgel biocatalytiques
AU2002366875B2 (en) * 2001-12-21 2008-08-21 Sense Proteomic Limited Probe for mass spectrometry
GB0202018D0 (en) * 2002-01-29 2002-03-13 Sense Proteomic Ltd Tag and method
US20060275855A1 (en) * 2002-02-21 2006-12-07 Blackburn Jonathan M Enzyme array and assay
GB0205910D0 (en) * 2002-03-13 2002-04-24 Sense Proteomic Ltd Arrays and methods
US20030228709A1 (en) * 2002-03-25 2003-12-11 Kozlowski Roland Zbignieiw Arrays
WO2004025244A2 (fr) 2002-09-16 2004-03-25 Sense Proteomic Limited Reseaux de proteines et leurs utilisations
JP2006506447A (ja) * 2002-10-25 2006-02-23 センス プロテオミック リミテッド Ｂｌｅ遺伝子によってコードされているタンパク質、及びブレオマイシン・ファミリー由来抗生物質の新規使用方法

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1543332A2 (fr) * 2002-09-16 2005-06-22 Sense Proteomic Limited Reseaux de proteines et leurs utilisations
JP2007510404A (ja) * 2003-10-15 2007-04-26 ジェネッサンス ファーマシューティカルズ，インコーポレイティド アルツハイマー病の発症年齢に関連するｎｔｒｋ１遺伝子マーカー
JP2007514417A (ja) * 2003-11-24 2007-06-07 ジェネッサンス ファーマシューティカルズ，インコーポレイティド アルツハイマー病の進行に関連するｎｔｒｋ１遺伝子マーカー
EP1561823A1 (fr) * 2004-02-04 2005-08-10 Biotez Berlin-Buch GmbH Procédé pour la détection des polymorphismes de nucléotide simple (SNP) des gênes du métabolisme des médicaments et dispositif pour l'utilisation correspondante
WO2006002525A1 (fr) * 2004-06-30 2006-01-12 Tm Bioscience Pgx, Inc. Methode de detection de mutations dans le gene codant pour le cytochrome p450-2c9
EP2993474A1 (fr) 2005-06-24 2016-03-09 Vermillion, Inc. Biomarqueurs pour le cancer de l'ovaire: la beta-2-microglobuline
EP2469279A1 (fr) 2006-03-11 2012-06-27 The Board Of Trustees Of The Leland Stanford Junior University Cystatin C, lysozyme et bêta-2 microglobuline en tant que biomarqueur d'une maladie des artères périphériques
US9228223B2 (en) 2007-02-12 2016-01-05 Codexis, Inc. Structure-activity relationships
WO2008123948A1 (fr) 2007-03-27 2008-10-16 Vermillion, Inc. Biomarqueurs pour cancer des ovaires
WO2009114292A1 (fr) 2008-03-10 2009-09-17 Lineagen, Inc. Signatures de biomarqueur de copd
EP3156925A2 (fr) 2008-03-10 2017-04-19 Lineagen, Inc. Signatures de biomarqueurs de la bpco
WO2012051519A2 (fr) 2010-10-14 2012-04-19 The Johns Hopkins University Biomarqueurs d'une lésion cérébrale
WO2012155134A2 (fr) 2011-05-12 2012-11-15 The Johns Hopkins University Réactifs de dosage pour kit de diagnostic de la neurogranine
WO2013106886A1 (fr) 2012-01-20 2013-07-25 Adelaide Research & Innovation Pty Ltd Biomarqueurs pour le cancer gastrique et leurs utilisations
WO2014100852A1 (fr) 2012-12-24 2014-07-03 Royal Melbourne Institute Of Technology Inhibition de la croissance du cancer et de métastase
WO2014182896A1 (fr) 2013-05-10 2014-11-13 Johns Hopkins University Compositions présentant une grande spécificité pour l'évaluation du cancer de l'ovaire
EP4286853A2 (fr) 2013-05-10 2023-12-06 Johns Hopkins University Compositions présentant une grande spécificité pour l'évaluation du cancer de l'ovaire
EP3757226A2 (fr) 2013-07-17 2020-12-30 The Johns Hopkins University Dosage de biomarqueurs multiprotéiniques pour la détection et l'issue de lésions cérébrales
US10914749B2 (en) 2014-09-19 2021-02-09 The Johns Hopkins University Biomarkers of cognitive dysfunction
US10222386B2 (en) 2014-09-19 2019-03-05 The Johns Hopkins University Biomarkers of congnitive dysfunction
US11041865B2 (en) 2015-02-20 2021-06-22 The Johns Hopkins University Biomarkers of myocardial injury
WO2017099829A1 (fr) 2015-12-11 2017-06-15 The General Hospital Corporation Compositions et procédés de traitement de glioblastome multiforme résistant aux médicaments
WO2019035005A1 (fr) 2017-08-16 2019-02-21 Medimmune, Llc Compositions et méthodes pour le traitement de la dermatite atopique et la sélection du traitement
WO2019236632A1 (fr) 2018-06-04 2019-12-12 Avon Products. Inc. Biomarqueurs protéiques permettant d'identifier et de traiter une peau vieillissante et des affections cutanées
US12116637B2 (en) 2020-07-24 2024-10-15 The Regents Of The University Of Michigan Compositions and methods for detecting and treating high grade subtypes of uterine cancer
WO2022072479A1 (fr) 2020-09-29 2022-04-07 The Johns Hopkins University Biomarqueurs protéomiques intégrés destinés à la détection d'un cancer agressif de la prostate
WO2023122723A1 (fr) 2021-12-23 2023-06-29 The Broad Institute, Inc. Panels et procédés de diagnostic et de traitement du cancer du poumon
WO2025106804A1 (fr) 2023-11-16 2025-05-22 Massachusetts Institute Of Technology Compositions et méthodes permettant la caractérisation d'un stress hépatocytaire
WO2025217069A1 (fr) 2024-04-08 2025-10-16 Beth Israel Deaconess Medical Center, Inc. Compositions et méthodes de traitement de la cardiotoxicité

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JP2009192547A (ja) 2009-08-27
EP1456668A2 (fr) 2004-09-15
CA2469403A1 (fr) 2003-06-12
GB2384239A (en) 2003-07-23
DE60219952D1 (de) 2007-06-14
JP4781628B2 (ja) 2011-09-28
AU2002352355A1 (en) 2003-06-17
AU2002352355B2 (en) 2008-04-03
EP1742062A3 (fr) 2008-09-10
EP1742062A2 (fr) 2007-01-10
EP1456668B1 (fr) 2007-05-02
WO2003048768A3 (fr) 2003-11-20
DK1456668T3 (da) 2007-09-10
US10870925B2 (en) 2020-12-22
JP2005512055A (ja) 2005-04-28
US20040002078A1 (en) 2004-01-01
US20180305840A1 (en) 2018-10-25
GB0228418D0 (en) 2003-01-08
ATE361471T1 (de) 2007-05-15
ES2289169T3 (es) 2008-02-01
DE60219952T2 (de) 2008-01-17

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